Herpes simplex virus (HSV) is a common and significant human pathogen that causes lifelong latent infection and a variety of diseases, ranging from cold sores and genital lesions to blindness and fatal encephalitis. Proteins within the tegument layer of HSV are released into the cell upon entry when the viral envelope fuses with the cell membrane. HSV capsids and capsid-associated tegument proteins then utilize the host cytoskeletal machinery for transport to the nucleus. We recently showed that incoming HSV employs the 26S proteasome machinery at a postpenetration step to initiate infection. ICP0 is a multi-functional viral protein that is synthesized by the infected cell with immediate-early kinetics. ICP0 has a zinc binding RING finger motif that confers E3 ubiquitin ligase activity. ICP0 is also a component of the virion tegument and is brought into the cell with the infecting viral particle. The functional role of tegument ICP0 has been largely undetermined. Our preliminary studies indicate a role for ICP0 in proteasome-dependent viral entry. The long-term goal of this project is to understand the virus-cell interactions that HSV uses to gain access to the host cell nucleus during the earliest stages of infection. The objective of this proposal is to determine the fate and function of tegument ICP0 in the context of pre-immediate early events in HSV infection. Our central hypothesis is that tegument ICP0 modulates the proteasome-dependent delivery of the viral nucleocapsid from the cell periphery to the nucleus. Two aims are proposed. In Specific Aim 1, the precise step in the entry process that is affected by ICP0 will be defined. The role of the RING finger domain of ICP0 in the proteasome-dependent transport of capsids will also be analyzed. In Specific Aim 2, the stability and subcellular localization of tegument ICP0 will be determined. The roles of the proteasome and of ICP0 functional domains in these processes will be defined. A combination of molecular, biochemical and cell biological approaches will be used to achieve these goals. These studies will widen our understanding of the mechanism for capsid targeting to the nucleus to initiate productive infection. By identifying new requirements for virus entry into the host, the results will increase our knowledge of the early steps of HSV infection and reveal targets for novel therapeutic intervention. PUBLIC HEALTH RELEVANCE: Herpes simplex virus is a common cause of infections and can cause serious complications such as neonatal infections and fatal encephalitis. The purpose of this research is to determine how the herpes simplex virus initiates infection in humans by studying the virus interaction with host cells. Greater understanding of the virus entry process may help to develop novel approaches to prevent herpes infections.